Research Of Artificial Infection Of Ibv And Quantitative Expression Analysis Of Inflammatory Activation-Related Factors

Inflammasome is a class of protein complexes that consist of a variety of proteins, including NLRP1(NLR family, Pyrin Domain Containing1), NLRP3, NLRP4and AIM2(Absent In Melanoma2), and NLRP3is closely associated with the recognition of virus infection. Studies have shown that inflammasome participates in the natural immune regulation by activating interleukin-1β and interleukin-18, activates the body to make response to infection and injury, which plays a very important role in innate immunity. Therefore, the study of inflammasome has an important significance in studying the inherent immunity and related diseases. In this study, we quantitatively detected the expression levels of four inflammasome-related genes after artificial infection of IBV (Infectious Bronchitis Virus), and analysed the expression change of inflammasome-related factors.The main contents are as follows.Firstly, we established the fluorescent quantitative detection methods of four inflammasome-related genes:NLRP3, Caspase-1, IL-1β and IL-18, with a high correlation coefficiency of standard curve, nearly100%of amplification efficiency and a good repeatability.Secondly, chicken tracheal rings in vitro were cultured and infected by the wild type strain GX-YL5and vaccine strain H120of infectious bronchitis virus (IBV). Then the tracheal rings were collected at day0,1,2,3,5,7, respectively. After total RNA was extracted, inflammasome-related genes were detected by fluorescent quantitative PCR. The results showed that NLRP3expression was up-regulated significantly (P<0.05), while the expression of Caspase-1, IL-1β and IL-18did not change significantly (P>0.05) in the IBV infection group compared with the H120group and the control group.Finally, chicks were infected by the wild type strain GX-YL5and vaccine strain H120of IBV, and trachea was collected at DPI day0,1,3,7,14,21, respectively. Then we extracted total protein and total RNA, and measured the caspase-1activity and quantitatively detected the expression levels of four inflammasome-related genes. The results showed that quantity of IBV in trachea increased after infection and reached to maximum at DPI day7, then gradually decreased. The activity of caspase-1and NLRP3expression were increasedat DPI day1-3, then decreased to a minimum at DPI day7, and then increased again. The expression of IL-1β and IL-18changed ups and downs at DPI day1-7, then reached to maximum in the later period of infection (IL-1β was at DPI day14, IL18was at DPI day21). In conclusion, the results showed that NLRP3inflammasome was activated in the early period of infected with IBV, leading to inflammation, and could up-regulate the expression of caspase-1, IL-1β, and IL-18, finally eliminated virus with acquired immunity synergetically.